Effects of Antimicrobial Peptide Supplementation on Immune Response and Immune Organ Index in Broiler Chicks

Antimicrobial peptides (AMPs) are small biological molecules weighing less than 10 kDa and containing less than 100 amino acids. They are found in all species, ranging from insects and plants to animals and have a broad spectrum of activity against bacteria, some viruses and fungi (Meyerholz and Ackermann, 2005). AMPs are produced by leucocytes, neutrophils and epithelial lining in the environmental interface of the gastrointestinal tract, urogenital tract, tracheobronchial tree and skin. In chicken, antimicrobial peptides have been isolated from bone marrow, tongue, trachea, bursa of Fabricius, brain, kidney, testicle, ovary, male and female reproductive tract, liver, the urogenital tract, gastrointestinal tract and lung (Lynn et al., 2004 and Ohashi et al., 2005).
       
Cathelicidins and defensins are important families of avian AMPs. Defensins are hydrophobic, cystine-rich, cationic antimicrobial peptides having the property of direct killing of microbes which includes bacteria, fungi, some of the enveloped viruses and most of the non-enveloped viruses and involved in immune signaling mechanisms (Selsted and Ouellette, 2005). Immunomodulatory properties of defensins have promoted the adaptive immunity by chemotaxis of T lymphocytes, monocytes, immature dendritic cells and mast cells (Niyonsaba et al., 2002) to the sites of inflammation. About 14 types of β-defensins have been discovered from different tissues in chicken so far. The expression of avian β-defensin 2 is moderate, 9 is weak and 13 is strong in the small intestine of chicken.
       
The misuse of antibiotics for growth promotion in poultry has led to bacterial antibiotic resistance and antibiotic residues in meat and egg (Marazuela and Bogialli, 2009). Hence, there is an urgent need to search for novel antibiotic substitutes for poultry having similar antimicrobial, immunomodulatory and growth promoting effects without inducing bacterial resistance and side effects. Probiotics, prebiotics, organic acids, phytobiotics, enzymes and antimicrobial peptides are a few of the potential alternatives to antibiotics that can be incorporated in feed. The objective of this study was to evaluate the effect of chicken intestinal AMP on humoral and cell-mediated immune response and immune organ index in broiler chicks.
 

Preparation of AMP
 
The AMPs were isolated from chicken small intestines as described by Ma et al., 2004. The duodenum portion was separated from the rest of the intestine. Mucosal scrapings of the duodenum were washed with cold sterile normal saline (0.85 % NaCl) and extracted in ice-cold aqueous 5% acetic acid (1:10 w / v) overnight. Later, the extracts were placed in boiling water for 10 minutes and cooled quickly. The supernatant was collected after centrifugation at 9000 rpm for 30 minutes at 4°C and pH was adjusted to 7.0 with 1 M NaOH. Purification of supernatant was done by size exclusion column chromatography (Bio Rad, USA). Each elution was analyzed by agarose diffusion assay (Lehrer et al., 1991) with E. coli (MTCC accession number: 452) and Salmonella (MTCC accession number: 98) as test organisms. The active fractions with potent antibacterial activity were collected and detected by SDS-PAGE and then outsourced for confirmation of molecular mass by LC-MS (Ashraf and Azad, 2017) at Sophisticated Analytical Instruments Facility, Indian Institute of Technology, Madras. The analysis of antimicrobial peptide by LC-MS revealed a molecular mass of 3.76 kDa which closely corresponded to the molecular mass of avian β-defensin 2 (3.9 kDa) with the NCBI Accession Number: AAB30585. The peptide-rich fractions were lyophilized and stored at 4°C until the feeding trial.
 
Animals and experimental design
 
The feeding trial was carried out for 5 weeks in the rabi  season of 2021 at the environmentally controlled poultry shed of Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai. A total of 90 one-day-old broiler chicks (Cobb 430) were weighed, wing banded and randomly allocated in a completely randomized design for 3 treatments, each of which had 3 replicates of 10 broiler chicks. The dietary treatments were a maize-soybean meal-based diet as a control (T1), control diet with 335 mg chlortetracycline (CTC) / kg (T2) and control diet with 100 mg AMP/kg (T3). Broiler chicks had ad libitum access to feed and water, with 23 hours of light per day. The average temperature during the biological trial period was 25.8°C. The standards of care used in the study were approved by Institutional Animal Ethics Committee (Approval Lr. No. 370/DFBS/IAEC/2021). Chicks were vaccinated as per the vaccination schedule given in Table 1.


 
Immunological assay
 
Sampling
 
Six birds from each treatment were randomly selected on the 21^st day and 35^th day, weighed and sacrificed. Immune organs were collected and weighed for immune organ index. Blood samples (2 ml) were collected for serum in plain vacutainers. Whole blood (4 ml) was collected in EDTA vacutainers for peripheral blood mononuclear cells (PBMCs) isolation on the 21^st day.

Serum antibody titre against infectious bursal disease and newcastle disease virus
 
Antibody titres were analyzed by ELISA using IDEXX IBD and NDV Ab Test kits respectively as per the manufacturer’s instructions. The viral antigen was pre-coated on 96 well plates. The serum (separated after centrifugation of blood samples at 3000 rpm for 5 minutes) was diluted to 500-fold with sample diluent provided in the kit and then 100 µl of diluted serum was added to the antigen pre-coated wells. 100 µl of both positive control (diluted chicken anti-NDV serum preserved with ProClin^TM 150) and negative control (diluted chicken serum non-reactive to NDV preserved with ProClin^TM 150) were also added to the wells and incubated for 30 minutes at room temperature (18-26°C). After incubation, the wells were washed thrice with approximately 350 µl of sterile distilled water. About, 100 µl of conjugate (HRP Goat anti-chicken antibody preserved with ProClin^TM 150) was added and the plate was incubated for 30 minutes at room temperature (18-26°C). After incubation, the wells were washed thrice with approximately 350 µl of sterile distilled water and 100 µl of TMB substrate was added to wells in darkness and left at room temperature for 15 minutes. The reaction was stopped by adding 100 µl of stop solution to each well. The OD values were recorded at 650 nm using an ELISA plate reader. Each sample was assayed in duplicate. Results were expressed in terms of log₁₀ titre values.
 
S/P= (Sample mean-NC)/(PC-NC)
 
Log₁₀ Titre= 1.09*(log₁₀ S/P) + 3.36
 
Where,
NC is negative control and PC is positive control.
 
Immune organ index
 
Spleen, bursa and thymus were collected and weighed to calculate the immune organ index as per the formula given below:
 
  

Gene expression of cytokines in Peripheral blood mononuclear cells (PBMCs)
 
Peripheral blood mononuclear cells were isolated as described by Kaiser et al. (2006) from the whole blood by density gradient centrifugation. The High-Capacity cDNA Reverse Transcription Kit (Takara Kit) was used for the synthesis of cDNA from RNA as per the manufacturer’s instructions. The cDNA synthesis cycle conditions were 37°C for 15 minutes followed by 85°C for 5 seconds. Real-time PCR analysis was performed for the expression of genes (IL-17A, IFN-α, IFN-γ, TGF-β, IL-10) with 16s rRNA gene as the housekeeping gene. Primers used for Real-Time PCR are presented in Table 2. Data were analyzed by the 2^-ΔΔCt method and normalized using the housekeeping gene expression level. Gene expression was measured by 7500 Fast Real-Time PCR (Applied Biosystem Inc, CA, USA) with SYBR PREMIX ex TAQTM (Perfect Real Time, Takara, Shiga, Japan). The thermal cyclic conditions used in Real-Time PCR were as follows: 95°C for 3 minutes, 40 cycles of 95°C for 30 seconds, 60°C for 15 seconds and 72°C for 35 seconds.


                                              
Statistical analysis
 
Experimental data were analyzed by one-way ANOVA using SPSS v.20.0 statistical package and means of different treatments were compared using Duncan’s multiple range test. The obtained results were expressed as mean± standard deviation.
 

The effect of supplementation of AMP on serum antibody titres and immune organ index are presented in Tables 3 and 4, respectively. Supplementation of AMP did not significantly alter the serum antibody titres against infectious bursal disease and Newcastle disease virus on the 21^st and 35^th days of the study. These findings were in agreement with the findings of Yoon et al., (2014) who reported no significant difference in serum antibody titres in weanling piglets fed with 60 mg/kg AMP. Maintenance of normal humoral response in the AMP supplemented group is attributed to the antiviral immunity provided by AMP and also the suppression of Th1 cells and stimulation of Th2 cells required for humoral immunity. No significant difference (P>0.05) was observed in the immune organ index among all the treatments. This might be due to the broad spectrum of activity of AMP which not only maintains the humoral immune response, but also reduces the proinflammatory cytokines and antigen-presenting cells activity resulting in restriction of the triggering mechanisms for increased immune cells production, thereby regulating the immune homeostasis. However, in contrast to the present findings, Bai et al., (2019) reported improved immune organ index and serum Newcastle disease antibody titre which would have been due to the supplementation of a higher dose of AMP (300-600 mg/kg).




       
The present study revealed for the first time the expression pattern of proinflammatory (IL-17A, IFN-α and IFN-γ) and anti-inflammatory (TGF-β and IL-10) cytokines in PBMCs of broiler chicken on dietary supplementation of AMP. PBMCs are considered potential immune cellular systems that harbor cytokines (Davila and De Los Rios, 2019). They are sensitized by circulating antigens that gain entry through the intestinal tract and modulate the cytokine expression depending on their concentration. Several studies disclose the importance of defensins in innate and humoral immune responses against a broad spectrum of microbes (Oppenheim et al., 2003; Yurong et al., 2006).  AMPs enhance the immune response by modulating the cytokine expression and activation of T and B cell production. In the present study, mRNA expression of proinflammatory cytokines viz., IL-17A, IFN-α and IFN-γ (Fig 1A, 1B and 1C, respectively) decreased and anti-inflammatory cytokine, IL-10 (Fig1E) increased. There was no significant change in the expression pattern of TGF-β (Fig 1D) as compared to the control group. IL-10, secreted by B lymphocytes regulates the B cell maturation and antibody production. Also, IL-10 suppresses the activity of natural killer cells and macrophages as well as downregulates the expression of pro-inflammatory cytokines, MHC II molecules and antigen-presenting cells activity thereby, playing an immunomodulatory role (Trinchieri, 2007). In this study, AMP supplementation improved the expression of IL-10 that led to the suppression of proinflammatory cytokines and Th1, while stimulated Th2 cell activity. TGF-β regulates cell proliferation, differentiation and growth. It has the ability to inhibit macrophage activation, T cell and B cell apoptosis as well as reduces the expression of inflammatory mediators such as TLR-4, IL-1, IL-6 and IFN-γ to maintain the immune homeostasis (Bickerstaff and Orosz, 2002). The current study results revealed no significant changes in the mRNA expression pattern of TGF-β gene and the basal level of gene expression required for regulation of cell cycle activity was maintained similar to the control group. IL-17A enhances the production of cytokines (IL-6, G-CSF, GM-CSF) chemokines (IL8, CXCL₁ and CXCL₅), neutrophil chemotaxis, antibody production and activation of T cells (Iwakura et al., 2008). In the present study, supplementation of AMP downregulated the mRNA expression of IL-17A mediated by IL-10. IFN-α (Type I interferon) exhibits a pleiotropic effect on the cells including antiviral immunity, innate immune response and modulating the cell fatality (survival/apoptosis). IFN-γ (Type II interferon) is a potential macrophage-activating factor and is also involved in the activation and regulation of innate and adaptive immune responses (Plachy et al., 1999). AMP supplementation downregulated the mRNA expression of IFN-α and IFN-γ in this study due to the antiviral defenses provided and enhanced cell protection by a reduction in the expression of interferon-stimulated genes, immunoproteasome activity and activation of other immune cells that prevent apoptosis.


 
 

Antimicrobial peptide supplementation had no significant (P>0.05) effect on immune organ index and serum antibody titres against infectious bursal disease and Newcastle disease virus measured on 21^st and 35^th days of the study. It significantly (P<0.01) downregulated the expression of pro-inflammatory cytokines (IL-17A, IFN-α, IFN-γ) and upregulated the expression of anti-inflammatory cytokine IL-10 on 21^st day of the study. Hence, it is concluded that supplementation of AMP, at a dietary concentration of 100 mg/kg enhanced the cell-mediated immune response of broiler chicks mediated through cytokines.
 

The authors thank Tamil Nadu Veterinary and Animal Sciences University for providing the necessary facilities to carry out the research work.
 

None.